Method for the detection or quantitation of an analyte using an analyte dependent enzyme activation system

ABSTRACT

The present invention concerns a method to catalyze reporter deposition to improve detection or quantitation of an analyte in a sample by amplifying the detector signal which comprises reacting an analyte dependent enzyme activation system with a conjugate consisting of a detectably labeled substrate specific for the enzyme system, said conjugate reacts with the analyte dependent enzyme activation system to form an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system. In another embodiment the invention concerns an assay for detecting or quantitating the presence or absence of an analyte in a sample using catalyzed reporter deposition to amplify the reporter signal.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/330,357,filed Mar. 29, 1989, now abandoned and U.S. Ser. No. 07/494,226, filedMar. 20, 1990 now abandoned.

FIELD OF THE INVENTION

This invention relates to assays and, more particularly, to catalyzingreporter deposition via an activated conjugate to amplify the detectorsignal thereby improving detection and/or quantitation of an analyte ina sample.

BACKGROUND OF THE INVENTION

The introduction of immunodiagnostic assays in the 1960s and 1970sgreatly increased the number of analytes amenable to precise andaccurate measurement. Radioimmunoassays (RIAs) and immunoradiometric(IRMA) assays utilize radioisotopic labeling of either an antibody or acompeting antigen to measure an analyte. Detection systems based onenzymes or fluorescent labels were developed as an alternative toisotopic detection systems. Enzyme based assays proved to be moresensitive, faster, and less dependent upon expensive, sophisticatedinstrumentation.

The need for diagnostic assays having simpler formats, increasedsensitivity with less dependence upon sophisticated and expensiveinstrumentation prompted investigators to try to harness the catalyticpower of enzymes to develop these newer assays.

D. L. Bates, Trends in Biotechnology, pages 204-209, Vol. 5 No. 7(1987), describes diagnostics which use a method of enzyme amplificationto develop more sensitive and simple immunoassays. In this method asecond enzyme system is coupled to the primary enzyme label, e.g., theprimary enzyme can be linked catalytically to an additional system suchas a substrate cycle or an enzyme cascade. Thus, the essence of enzymeamplification according to Bates is the coupling of catalytic processeswherein an enzyme is modulated by the action of a second enzyme, eitherby direct modification or by interaction with the product of thecontrolling enzyme.

U.S. Pat. No. 4,668,621, issued to Doellgast on May 26, 1987, describesapplication of an enzyme-linked coagulation assay (ELCA) to develop anamplified immunoassay using the clotting cascade to enhance sensitivityof detection of immune complexes. The process involves clot formationdue to thrombin activated fibrin formation from insolubilized fibrinogenand labeled solubilized fibrinogen. Amplification of the amount ofreportable ligand attached to solid phase is obtained only by combininguse of clotting factor conjugates with subsequent coagulation cascadereactions. One of the disadvantages of this system is that it can onlybe used to measure the presence of materials which modulate the activityof one or more of the blood clotting factors. Another disadvantage isthat the primary enzyme, thrombin, cannot be immobilized or coupled to areporter or a member of a specific binding pair.

U.S. Pat. No. 4,463,090, issued to Harris on Jul. 31, 1984, describes acascade amplification immunoassay requiring a combination of at leasttwo sequential catalyses wherein a first enzyme activates a secondenzyme which in turn acts upon the substrate.

Another amplification system is described in U.S. Pat. No. 4,598,042,issued to Self on Jul 1, 1986, and U.K. Patent Application No. 2,059,421which was published on Apr. 23, 1981, which disclose an immunoassayusing an enzyme label to produce directly or indirectly a substance thatis capable of influencing a catalytic event without itself beingconsumed during the catalytic event. More specifically, a primary enzymesystem produces or removes a substance capable of modulating a secondaryenzyme system which results in amplification. The enzyme systems useunconjugated enzymes to avoid the tendency to inactivate certain enzymeson conjugation.

European Patent Application Publication No. 123,265 which was publishedon Oct. 31, 1984, describes another cascade amplification immunoassaywherein a zymogen-derived-enzyme is coupled to a zymogen-to-enzymecascade reaction sequence to obtain multiple stages of amplification inproducing detectable marker material used to quantify analyte amount.

European Patent Application Publication No. 144,744, published Jun. 19,1985, describes a specific binding assay based on enzyme cascadeamplification wherein the label component employed in the detectantreagent is a participant in or a modulator of an enzyme cascade reactionwherein a first enzyme acts on a first substrate to product a secondenzyme. The production of the second enzyme can be followed or thesecond enzyme can act on a second substrate to produce a third enzyme.

Similarly, U.S. Pat. No. 4,318,980, issued to Boguslaski et al. on Mar.9, 1982, describes a heterogenous specific binding assay using aconjugate formed of a specific binding substance coupled to thereactant, i.e., an enzymatic reactant. The ability of the reactant toparticipate in the monitoring reaction to detect the presence of analyteis altered by the presence of the ligand in the medium. Thus, theconjugate in its free state is more active in the monitoring reactionthan in its bound state.

A heterogenous specific binding assay using enzyme amplification isdescribed in British Patent Application No. 1,401,297 which waspublished on Jul. 30, 1975 and U.S. Pat. No. 4,376,825, issued toRubenstein et al. on Mar. 15, 1983. Amplification is achieved by bondingthe compound to be assayed or a counterfeit of it to an enzyme. Theresulting enzyme-bound-ligand competes with free ligand for specificreceptor sites. When the enzyme-bound ligand is displaced by the freeligand the enzyme is then free to react with a large number number ofsubstrate molecules and the concentration of the remaining substrate orof the product can be measured. PCT International Publication No. WO81/00725 which was published on Mar. 19, 1981 describes a method ofdetermining a substrate in a sample which comprises converting thesubstrate to a product in a first stage of a cyclic reaction sequenceand converting the product back to the substrate in a second reactionstage of the cyclic reaction sequence. At least one of the first andsecond reaction stages is enzyme catalyzed.

PCT Application having International Publication Number WO 84/02193,which was published on Jun. 7, 1984, describes a chromgenic supportimmunoassay wherein the analyte is contacted with an enzyme-labeledantibody and in which the signal generated by the reaction of the enzymewith its substrate is concentrated on an active support.

European Patent Application Publication No. 181,762, published on May21, 1986, describes a method to determine enzymatic activity in a liquidsample by particle agglutination or inhibition of particleagglutination.

Substrate/cofactor cycling is another example of amplification which isbased on the cycling of a cofactor or substrate which is generated bythe primary enzyme label. The primary enzyme converts the primarysubstrate to an active form which can be cycled by two enzymes of theamplifier cycle. These two enzymes are provided in high concentrationand are poised to turn over high concentrations of substrate but areprevented from so doing until the cycling substrate is formed. Theproduct of the primary enzyme is a catalytic activator of the amplifiercycle which responds in proportion to the concentration of substrate andhence the concentration of the enzyme label.

In the early sixties, Lowry et al., Journal of Biological Chemistry,pages 2746-2755, Vol. 236, No. 10 (Oct. 1961), described the measurementof pyridine nucleotides by enzymatic cycling in which the coenzyme to bedetermined was made to amplify an enzymatic dismutation between twosubstrates.

A more complex substrate cycling system is described in U.S. Pat. No.4,745,054, issued to Rabin et al. on May 17, 1988. The Rabin systeminvolves using a small enzymically inactive peptide fragment of anenzyme as a label and conjugated with the complementary fragment to forman enzyme which catalyzes a primary reaction whose product is, or leadsto, an essential coenzyme or prosthetic group for a second enzyme whichcatalyzes a secondary reaction leading to a detectable result indicatingthe presence of analyte.

Vary et al., Clinical Chemistry, pages 1696-1701, Vol. 32 (1986)describe an amplification method suited to nucleic acids. This is thestrand displacement assay which uses the unique ability of apolynucleotide to act as a substrate label which can be released by aphosphorylase.

SUMMARY OF THE INVENTION

The present invention concerns a method to catalyze reporter depositionto improve detection or quantitation of an analyte in a sample byamplifying the detector signal which comprises reacting an analytedependent enzyme activation system with a conjugate consisting of adetectably labeled substrate specific for the enzyme system, saidconjugate reacts with the analyte dependent enzyme activation system toproduce an activated conjugate which deposits substantially whereverreceptor for the activated conjugate is immobilized, said receptor notbeing reactive with the analyte dependent enzyme activation system.

In another embodiment the invention concerns an assay for detecting orquantitating the presence or absence of an analyte in a sample usingcatalyzed reporter deposition to amplify the reporter signal.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a graph comparing results of an HSV antigen assay run with andwithout catalyzed reporter deposition.

FIG. 2 is a graph comparing results of an HIV p24 core antigen assayusing conjugate concentrations of 0.2, 0.4, and 0.8 μl/ml (Amp 1, 2, and3, respectively) "HRP" represents a non-amplified assay wherein thedetector antibody was directly labeled with HRP. "Biotin" indicatesanother non-amplified assay wherein the detector antibody was conjugatedto biotin and detected with HRP labeled streptavidin.

FIG. 3 is a graph of a mouse IgG assay run using an HRP ADEAS tocatalyze deposition of biotin-tyramine which was detected withstreptavidin-HRP (HRP-Amp HRP) or with streptavidin-AP (HRP-Amp AP). Theassay was also run using only HRP labeled detector antibody or APlabeled detector antibody.

FIG. 4 presents two graphs comparing results obtained from a Du Pont HIVp24 antigen ELISA run with and without using catalyzed reporterdeposition to amplify reporter signal.

FIG. 5 is a graph comparing results of a mouse IgG assay withoutcatalyzed reporter deposition (HRP) and with catalyzed reporterdeposition (HRP-β-Gal).

FIG. 6 depicts the synthesis of7-(β-galactosyl)-4-methylcoumarin-3-acetamidobiocytin.

FIG. 7 is a graph comparing the results obtained in detecting aβ-galactosidase ADEAS using a conventional, non-amplified assay formatversus results obtained in detecting a β-galactosidase ADEAS usingcatalyzed reporter deposition to amplify the detector signal.

DETAILED DESCRIPTION OF THE INVENTION

The term analyte dependent enzyme activation system (ADEAS) refers to anenzyme system wherein (i) at least one enzyme is coupled, in any mannerknown to those skilled in the art, to a member of a specific bindingpair, or (ii) the enzyme need not be coupled to a member of a specificbinding pair when it is the analyte. The enzyme, either by itself or inconnection with a second enzyme, catalyzes the formation of an activatedconjugate which then is deposited wherever a receptor for the activatedconjugate is immobilized.

The term amplification as used herein means amplification of reportersignal due to deposition of a conjugate activated by an ADEAS.

The term conjugate means a detectably labeled substrate specific for theADEAS whether it be a single enzyme ADEAS or multi-enzyme ADEAS. Thesubstrate must have at least one component but is not limited to such.For example, the substrate can consist of two components. One componentcontains the binding site for the receptor and is detectably labeled.The other component is a constituent which prevents or interferes withbinding to the receptor until such time as the ADEAS primes theconjugate as is discussed below. Another example of a conjugate isbiotin-tyramine wherein tyramine is the substrate portion and biotinconstitutes the detectable label as described below. Conjugates aredescribed in greater detail below as well.

The term detectably labeled means that the substrate can be coupled toeither a reporter or to an unlabeled first member of a specific bindingpair provided that the reporter introduces a different moiety to thesubstrate as is discussed below. When the substrate is coupled to anunlabeled member of a specific binding pair, following deposition, thesubstrate-specific binding partner complex is reacted with the secondmember of the binding pair which is coupled to a reporter. Alternately,the substratespecific binding partner complex can be pre-reacted withthe detectably labeled other member of the specific binding pair priorto deposition.

The term deposition means directed binding of an activated conjugate tothe receptor which results from the formation of a specific binding pairinteraction as described below.

The term receptor means a site which will bind to the activatedconjugate through the formation of a specific binding pair interactionas described below.

The term activated conjugate means that the conjugate has been primed bythe ADEAS to bind with the receptor.

One of the unique features of this invention is the analyte dependentenzyme activation system which catalyzes deposition of conjugate byconverting the substrate portion of the conjugate to an activated formwhich is deposited wherever a specific receptor for the activatedconjugate is immobilized. The ADEAS does not utilize enzyme cascadereactions or enzyme cycling to effect amplification. Rather, it useseither a single enzyme or combination of enzymes to activate theconjugate. Deposition of conjugate occurs only if the analyte andanalyte dependent enzyme activation system, which can be the same if theanalyte is an enzyme, for example in the detection of an enzyme such asalkaline phosphatase, or different, have been reacted and a receptor, asdescribed below, is immobilized to bind the activated conjugate. Thus,the ADEAS, conjugate, and receptor are chosen to form an operationaltrio.

The following is one embodiment of a single enzyme ADEAS system appliedto a forward sandwich immunoassay format: the test sample containing theanalyte is reacted with an immobilized capture reagent, such as anantibody; excess reagents are washed off; the immobilized captureantibody-analyte complex is reacted with an ADEAS, such as a secondantibody specific for the analyte which has been coupled to an enzyme,e.g. horseradish peroxidase (HRP), alkaline phosphatase (AP), etc. TheADEAS will bind only if the analyte has been bound by the capturereagent. Otherwise the reagents will be washed off. Coupling of theenzyme to a specific binding partner does not affect the enzyme'sability to react with the substrate portion of the conjugate. Whenconjugate such as biotin-tyramine is added to the immobilized captureantibody-analyte-second antibody-enzyme complex, the enzyme reacts withthe substrate portion of the conjugate, in this case, with the tyramineportion of the conjugate, converting it to an active form which willbind to an immobilized receptor which is either endogenous or exogenousto the assay system. The amount of conjugate deposited will be afunction of immobilized ADEAS. Deposited conjugate such asbiotin-tyramine can then be detected by reacting with streptavidin-HRPand orthophenylenediamine. If the conjugate is fluorescein-tyramine thenthe deposited conjugate can be detected directly, or following reactionwith a labeled anti-fluorescein antibody.

Thus, the ADEAS is used to catalyze the deposition of detectably labeledsubstrate (the conjugate) to generate additional signal. The ADEAS isdetected directly as part of the overall signal when the enzymecomponent of the ADEAS is the same as the enzyme used as the reporter.FIG. 3 illustrates this situation as well as the situation where anADEAS enzyme component and reporter enzyme are different and thus, theADEAS enzyme component is not detected directly as part of the overallsignal.

A multi-enzyme ADEAS immunoassay format would involve a similarapproach. For example, the ADEAS can be an antibody coupled to an enzymesuch as neuraminidase which is reacted with immobilized capture antibodyand analyte to form an immobilized capture antibody-analyte-secondantibody-neuraminidase complex. A second enzyme such as β-galactosidaseis added together with the conjugate. The conjugate can be a detectablylabeled moiety containing a sialyl-galactosyl glycoside. Neuraminidasereleases the terminal sialic acid residue which then enablesβ-galactosidase to remove the galactose group. Without the release ofthe terminal sialic acid residue, the β-galactosidase cannot remove thegalactose group. Once deglycosylation is complete, the activatedconjugate deposits wherever receptors for the activated conjugate areimmobilized.

The instant invention is surprising and unexpected because amplificationof reporter signal is obtained via deposited activated conjugate withoutusing cascade mechanisms or enzyme cycling. The ADEAS reacts with theconjugate to form an activated conjugate which will bind withimmobilized receptor specific for the activated conjugate. The amountsof receptor and activated conjugate are in excess of the amount of ADEASimmobilized.

The choice of an ADEAS is governed by the ability of the enzyme orenzymes to convert a conjugate to an activated form which will bind toan immobilized receptor whether endogenous or exogenous. Accordingly, adetailed knowledge of catalytic properties of each specific enzyme isneeded in order to properly design the substrate and receptor. Otherimportant factors include availability of the enzyme or enzymes,relative ease or difficulty of coupling it to the member of a specificbinding pair, stability of the enzyme or enzymes as well as thestability of the conjugate and the receptor. In some cases, an ADEAS canbe purchased, depending on the assay format.

Enzymes suitable for use in an ADEAS include hydrolases, lyases,oxidoreductases, transferases isomerases and ligases. There can bementioned peroxidases, oxidases, phosphatases, esterases andglycosidases. Specific examples include alkaline phosphatase, lipases,beta-galactosidase and horseradish peroxidase.

Members of specific binding pairs suitable for use in practicing theinvention can be of the immune or nonimmune type. Immune specificbinding pairs are exemplified by antigen/antibody systems orhapten/antihapten systems. The antibody member, whether polyclonal,monoclonal or an immunoreactive fragment thereof, of the binding paircan be produced by customary methods familiar to those skilled in theart. The terms immunoreactive antibody fragment or immunoreactivefragment mean fragments which contain the binding region of theantibody. Such fragments may be Fab-type fragments which are defined asfragments devoid of the Fc portion, e.g., Fab, Fab' and F(ab')₂fragments, or may be so-called "half-molecule" fragments obtained byreductive cleavage of the disulfide bonds connecting the heavy chaincomponents of the intact antibody. If the antigen member of the specificbinding pair is not immunogenic, e.g., a hapten, it can be covalentlycoupled to a carrier protein to render it immunogenic.

Non-immune binding pairs include systems wherein the two componentsshare a natural affinity for each other but are not antibodies.Exemplary non-immune binding pairs are biotin-avidin orbiotin-streptavidin, folic acid-folate binding protein, complementaryprobe nucleic acids, etc. Also included are non-immune binding pairswhich form a covalent bond with each other. Exemplary covalent bindingpairs include sulfhydryl reactive groups such as maleimides andhaloacetyl derivatives and amine reactive groups such asisothiocyanates, succinimidyl esters, sulfonyl halides, and coupler dyessuch as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and3-(dimethyl-amino)benzoic acid (DMAB), etc.

Suitable supports used in assays include synthetic polymer supports,such as polystyrene, polypropylene, substituted polystyrene, e.g.,aminated or carboxylated polystyrene; polyacrylamides; polyamides;polyvinylchloride, etc.; glass beads; agarose; nitrocellulose; nylon;polyvinylidenedifluoride; surface-modified nylon, etc.

Another important component of the invention is the conjugate, i.e., adetectably labeled substrate which must be specific for the ADEAS. Aswas stated above, when the conjugate reacts with the ADEAS, the enzymeor enzymes catalyze formation of an activated conjugate which bindswherever a receptor is immobilized whether exogenous or endogenous. Theactivated conjugate binds to the receptor via a specific binding pairinteraction as described above. An immobilized exogenous receptor meansa receptor which does not originate within the assay. It must beimmobilized on the surface of a support prior to adding the conjugate tothe reaction mixture. An endogenous receptor means a receptor whichoriginates within the assay and does not require immobilization prior toadding the conjugate because the receptor is immobilized within theassay system.

For example, when an HRP ADEAS (HRP coupled to a member of a specificbinding pair) is reacted with conjugate containing a phenolic substrate,an activated phenolic substrate is produced. It is believed that theactivated phenolic substrate binds to electron rich moieties such astyrosine and tryptophan present in the proteins on the solid support.However, if a different conjugate is used, such as a labeled MBTH whichis discussed below, a receptor, such as an analog of DMAB, must beimmobilized prior to addition of conjugate.

Another embodiment involves reacting a conjugate which becomesphosphorylated by an ADEAS. The activated (phosphorylated) conjugate canthen react with an antibody specific for the activated conjugate.

In still another variation, an ADEAS can be reacted with a conjugateconsisting of a component which when activated will bind to a receptorand which is coupled to a component having a thiol reactive group suchas a maleimide. The deposited maleimide moiety can then be detected byreacting with a sulfhydryl-containing reporter which can be endogenousto the reporter, e.g., beta-galactosidase, or the sulfhydryl groups canbe added to reporters such as HRP or AP using thiolating reagents suchas N-succinimidyl-S-acetylthioacetate (SATA), S-acetylmercaptosuccinicanhydride (SAMSA), or succinimidyl-3-(acetylthio)-propionate (SATP).

Alternatively, the substrate can be coupled to a protected sulfhydrylcontaining group and this can be used as the conjugate. After binding tothe receptor, this can be deprotected using conventional techniquesknown to those skilled in the art. Detection can be effected using areporter having a thiol reactive group such as maleimide-HRP oriodoacetyl-HRP.

Another alternative is to use a conjugate wherein the substrate has twocomponents as described above, a detectably labeled first componentwhich will bind to the receptor after the second component has beenactivated or removed by the ADEAS.

Other small organic molecule/receptor combinations which are suitable topractice the invention include haptens/antibodies, sugars andoligosaccharides/lectins, biotin and avidin/streptavidin.

As is shown in Table 1, a number of receptors are available. The choiceof a receptor will depend upon the conjugate selected.

The optimal concentration of conjugate is determined according to theprocedure explained in Example 1. Optimal concentrations will varydepending upon enzyme used in the ADEAS and substrate selected toproduce conjugate.

Conjugate can be synthesized using conventional coupling and labelingtechniques. Substrate choice will depend upon the ADEAS selected. Toreiterate, detailed knowledge is required of the catalytic properties ofeach specific enzyme in order to properly design a useful syntheticsubstrate and, if necessary, a receptor.

A wide variety of reporters are available for coupling to the substrateto produce the conjugate or to couple to a member of a specific bindingpair. As was discussed above reporter should introduce a differentmoiety to the substrate. Reporters can be a radioactive isotope, suchas, ¹²⁵ I, enzymes, fluorogenic, chemiluminescent, or electrochemicalmaterials. Internally labeled reporters (e.g., tritium or other suchradionuclides) which do not introduce a different moiety to thesubstrate are not contemplated for practicing the invention.

Examples of reporter enzymes which can be used to practice the inventioninclude hydrolases, lyases, oxidoreductases, transferases, isomerasesand ligases. Some preferred examples are phosphatases, esterases,glycosidases and peroxidases. Specific examples include alkalinephosphatase, lipases, beta-galactosidase and horseradish peroxidase. Aswas noted above, if an enzyme is used as a reporter, it can be the sameas or different from the enzyme or enzymes used in the ADEAS. Theinstant invention can be used to catalyze deposition of aradioisotopically labeled conjugate or an enzymelabeled conjugate, etc.

Another embodiment of the forward sandwich immunoassay described abovewould involve reacting a capture-antibody-analyte-second antibodycomplex with an ADEAS consisting of an anti-antibody coupled to anenzyme such as HRP or AP. The anti-antibody would bind an epitope on thesecond antibody.

This invention is not limited to sandwich immunoassays. It is applicableto a wide variety of assay formats, for example, nucleic acidhybridization assays for both RNA and DNA.

To further illustrate the invention, examples of single and multi-enzymeADEAS', conjugates, receptors, and receptor types are presented in Table1 below.

                                      TABLE 1                                     __________________________________________________________________________              ADEAS       CONJUGATE.sup.2                                                                             RECEPTOR                                  Class   Enzyme    Substrate    Class  Type                                    __________________________________________________________________________    Single Enzyme                                                                         HRP.sup.1 Substituted phenols,                                                                       Endogenous                                                                           Phenols; electron                                         e.g., tyramine      rich moieties                           Single Enzyme                                                                         HRP.sup.1 MBTH         Exogenous                                                                            DMAB                                    Single Enzyme                                                                         β-Galactosidase                                                                    β-Galactopyranosyl-                                                                   Exogenous                                                                            Antibody to                                               glycoside, e.g., of deglycosylated                                            fluorescein, coumarin,                                                                            moiety, e.g.,                                             etc.                anti-fluorescein;                                                             anti-coumarin                           Single Enzyme                                                                         AP        NADP         Exogenous                                                                            NAD binding                                                                   proteins                                Single Enzyme                                                                         AP        Substituted phosphates,                                                                    Exogenous                                                                            Antibody to                                               e.g., nitrophenyl   dephosphorylated                                          phosphate           product, e.g.,                                                                anti-nitro-phenol                       Single Enzyme                                                                         AP        Phosphorylated                                                                             Exogenous                                                                            Avidin;                                                   biotin              streptavidin                            Multi-Enzyme                                                                          AP and HRP.sup.1                                                                        Phosphorylated substi-                                                                     Endogenous                                                                           Phenols; electron                                         tuted phenols, e.g.,                                                                              rich moieties                                             tyrosine phosphate                                          Multi-Enzyme                                                                          Neuraminidase and                                                                       Sialyl-β-galactopyranosyl-                                                            Exogenous                                                                            Antibody to deglyco-                            β-galactosidase                                                                    glycoside of coumarin                                                                             sylated moiety, e.g.,                                                         anti-coumarin                           __________________________________________________________________________     .sup.1 HRP requires the presence of H.sub.2 O.sub.2.                          .sup.2 Label can be a reporter or member of a specific binding pair.     

In the AP/HRP multi-enzyme ADEAS described above, the conjugate must bedephosphorylated before it will react with HRP; and in theβ-gal/neuraminidase multienzyme ADEAS, the conjugate must bedesialylated before it will react with β-gal.

It should be clear to those skilled in the art that a large number ofvariations are possible and all these variations fall within the scopeof the invention.

The following examples are intended to illustrate the invention. Unlessotherwise indicated, 100 μl of all reagents were used for the EIA stripassays. The one exception was that 200 μl of blocking buffer was used.

EXAMPLE 1 Preparation of Conjugates and Optimization of ConjugateConcentration

Para-hydroxyphenylpropionyl biocytin (HPPB) was prepared by mixing asolution of p-hydroxyphenylpropionic acid-N-hydroxysuccinimide ester (50mg [0.2 mMol]/2 ml dimethyl sulfoxide) with biocytin (70.75 mg [0.2mMol]/2 ml 0.1M NaHCO₃) overnight at room temperature (RT).Biotin-tyramine (BT) was prepared by mixing a solution of tyramine (40mg [0.3 mMol]/1 ml dimethyl sulfoxide) with biotin-N-hydroxysuccinimideester (100 mg [0.3 mMol]/1 ml dimethyl sulfoxide) overnight at RT. Thesolutions of HPPB and BT were used as is. The calculated concentrationswere 26 mg/ml for HPPB and 55 mg/ml for BT.

Polystyrene EIA strips (NUNC) were coated with polyclonal anti-HerpesSimplex Virus (HSV) antibody (Dako, Carpenteria, CA) in 0.1M carbonatebuffer pH 9.6 overnight at 4° C., and then blocked with 2% bovine serumalbumin (BSA) in carbonate buffer and then washed with 10 mM phosphatebuffered saline, 0.05% Tween 20, pH 7.4 (PBST). A dilution of HSVantigen in 1% BSA, 10 mM phosphate buffered saline, 0.05% Tween 20 pH7.4 (BSAPBST), or buffer without antigen, was incubated for 1 hour at37° C. The dilution was sufficient to obtain the optical densities inthe range reported in Table 1. It was washed with PBST. The analytedependent enzyme activation system consisted of HRP coupled to anti-HSV(HRP ADEAS) which was purchased from Dako. The HRP ADEAS was added andincubated for 30 min. at RT and was washed with PBST. Variousconcentrations of HPPB or BT as set forth in Table 1 below, were addedin 50 mM tris-HCl, 0.01% H₂ O₂, pH 8.0, for 15 min. at RT. After washingwith PBST, streptavidin-HRP was added and incubated for 15 min. at RT toreact with deposited biotins. The plate was then washed with PBST. AnHRP substrate, o-phenylenediamine (OPD), was added, incubated for 30min. at RT, and stopped with 4N H₂ SO₄. Optical densities at 490 nm wererecorded on a microtiter plate reader.

Results

Results are presented in Table 2. Column 1 presents the variousconcentrations in μl/ml of HPPB or BT. Columns 2 and 3 present theoptical densities recorded as a function of HPPB concentration. Columns4 and 5 present the results obtained using BT.

HPPB and BT were converted to activated forms by HRP ADEAS. Catalyzedreporter deposition was achieved without immobilizing a receptor.

In choosing the optimal concentration, one must look at both themagnitude of signal amplification as well as the signal to noise ratio.With this in mind, the optimal concentration of HPPB was 20 μl/ml(approximately 0.5 mg/ml), and that of BT, was about 0.3 μ/ml(approximately 16 μg/ml).

                  TABLE 2                                                         ______________________________________                                        Absorbance 490 nm                                                             Conc. HPPB                                                                              HPPB CONJUGATE  BT CONJUGATE                                        or BT              Buffer            Buffer                                   (μl/ml)                                                                              HSV      (w/o Ag)*  HSV    (w/o Ag)                                 ______________________________________                                        0         0.079    0.031      0.079  0.031                                    20        1.155    0.181      0.700  0.165                                    10        0.904    0.140      --     --                                       5         0.499    0.120      2.060  0.430                                    2.5       0.177    0.063      --     --                                       1.25      0.113    0.062      2.230  0.502                                    0.625     0.103    0.048      --     --                                       0.313     --       --         1.880  0.169                                    0.078     --       --         0.263  0.051                                    0.020     --       --         0.090  0.040                                    ______________________________________                                         *w/o Ag = without antigen                                                

EXAMPLE 2 Amplification of Detector Signal In HSV Assay Using CatalyzedReporter Deposition

Anti-HSV coated EIA strips were prepared as described in Example 1. A1:100 dilution of HSV antigen was prepared and serially four-folddiluted. These dilutons of HSV were incubated for 2 hours at 37° C. withthe anti-HSV coated EIA strips. Excess reagent was washed off with PBST.The ADEAS was the same as that described in Example 1 above. It wasadded to the anti-HSV coated EIA strips containing the anti-HSV-HSVcomplex and incubated for 30 min. at RT and then washed with PBST. 20μl/ml of HPPB conjugate as determined in Example 1 was added in 50 mMtris-HCl, 0.01% H₂ O₂, pH 8.0, and was incubated for 15 min. at RT andthen washed with PBST. Deposited biotins were reacted withstreptavidin-HRP (SA-HRP) for 15 min. at RT followed by washing withPBST. The substrate, OPD, was added and incubated 30 min. at RT, stoppedwith 4N H₂ SO₄, and the absorbance at 490 nm was recorded on amicrotiter plate reader.

Non-amplified assays were run in which (a) no HPPB and no SA-HRP wereused; (b) HPPB was used without SA-HRP; (c) SA-HRP was used withoutHPPB.

Results

The results shown in FIG. 1 demonstrate that (a) catalyzed deposition ofreporter was obtained and (b) both the conjugate and SA-HRP were neededfor detection because the conjugate contained an unlabeled member of aspecific binding pair.

Results for the non-amplified assay (no HPPB, no SA-HRP) were plotted.The results for the other assays were not plotted because the additionalplots would overlap with the non-amplified results already plotted.

EXAMPLE 3 Amplification of Detector Signal in HIV p24 Assay UsingCatalyzed Reporter Deposition: Effect of Conjugate Concentration

Polystyrene EIA strips (NUNC) were coated with rabbit anti-HIV p24antibodies in 0.1M carbonate buffer, pH 9.6, overnight at 4° C., andthen blocked with 2% BSA in carbonate buffer followed by washing withPBST. HIV (human immunodeficiency virus) antigen was incubated for 2hours at 37° C. (concentrations are indicated in FIG. 2). The plate wasthen washed with PBST. A rabbit anti-HIV p24-HRP analyte dependentenzyme activation system was then incubated for 2 hours at 37° C., andwashed with PBST. Various concentrations of BT conjugate, (0.2, 0.4, and0.8 μl/ml) in 0.1M borate buffer, 0.01% H₂ O₂, pH 8.5, were incubatedfor 15 min. at RT followed by washing with PBST. Then streptavidin-HRPwas incubated for 15 min. at RT.

As a comparison, a biotinylated anti-HIV p24 antibody was used, anddetected with streptavidin-HRP. OPD was added and incubated for 30minutes, stopped with 4N H₂ SO₄, and optical densities at 490 nm wererecorded on a microtiter plate reader.

Results

The results are shown in FIG. 2 where Amp 1, Amp 2, and Amp 3 refer toBT at concentrations of 0.2, 0.4, and 0.8 μl/ml respectively. Differentlevels of amplification were achieved using catalyzed reporterdeposition depending on the concentration of conjugate.

FIG. 2 also presents results for a non-amplified assay using abiotinylated antibody/SA-HRP detection system (Biotin) and anon-amplified assay wherein anti-HIV p24 detector was directly labeledwith HRP (HRP). The results obtained using the anti-HIV p24-HRP detectorwere inferior compared to the significant increase in detector signalobtained using catalyzed reporter deposition.

Depending upon the concentration of conjugate, signals as good, andgreater, as those obtained with the biotinylated antibody were obtainedusing the catalyzed reporter deposition method of the instant invention.Best results were obtained using conjugate concentration near theoptimal amount as was determined in Example 1.

EXAMPLE 4 Preparation and Characterization of Biotin Tyramine

Synthesis of biotin-tyramine: a solution of biotin-N-hydroxysuccinimide,170 mg (0.5 mMoles), and tyramine (recrystallized from water), 68.5 mg(0.5 mMoles), in 25 ml dimethylformamide was treated with 10 ml of 1Mtriethylammonium bicarbonate, pH 7.5, and then heated at 50° C. for 3hours.

Isolation: the solution was concentrated to dryness on a rotaryevaporator, and the residue was recrystallized from water, with a yieldof 72%.

Characterization: the melting point was determined to be 192°-193° C.

EXAMPLE 5 Amplification of Detector Signal In A Mouse IgG Assay UsingCatalyzed Reporter Deposition

Polystyrene EIA strips (NUNC) were coated with goat anti-mouse IgG (Fcfragment specific) antibody (ICN) in 0.1M carbonate buffer pH 9.6,overnight at RT. They were then blocked with 2% BSA in carbonate bufferand washed with PBST. Dilutions of mouse IgG in BSA-PBST were incubatedin the wells for 1 hour at 37° C. followed by washing with PBST.Concentrations are set forth in FIG. 3. Goat anti-mouse IgG-HRP (HRPADEAS) and goat anti-mouse IgG-Alkaline Phosphatase (AP ADEAS)(Boehringer Mannheim) were diluted as recommended by the manufacturerand incubated for 1 hour at 37° C. Assays were run with and withoutcatalyzed reporter deposition. The AP ADEAS was not used to catalyzereporter deposition in this experiment.

For catalyzed reporter deposition using the HRP ADEAS, a 1 mg/ml stocksolution of biotin tyramine (as described in Example 4) in dimethylsulfoxide was prepared, and then added to a 0.1M borate buffer, pH 8 5,0.01% H₂ O₂, at 10 μl/ml (10 μg/ml biotin tyramine) and incubated for 15min. at RT. The plate was then washed with PBST. Streptavidin-HRP (forHRP-Amp HRP), or streptavidin-Alkaline Phosphatase (for HRP-Amp AlkPhos) were incubated for 15 min. at RT and the plate was washed withPBST. Spectrophotometric detection was achieved after the addition ofOPD (for HRP), or p-nitrophenyl phosphate (for AP) for 15 min. at RT.Reactions were stopped by the addition of 4N H₂ SO₄ (HRP/OPD), or 1NNaOH (Alk Phos/pNPP). Optical densities, at 490 nm for HRP/OPD and 405nm for Alk Phos/pNPP, were recorded on a microtiter plate reader.

Results

The results are shown in FIG. 3. As is apparent, one can achieve signalamplification with a concomitant lower detection limit by allowing theHRP ADEAS to catalyze deposition of an activated BT conjugate followedby detection with streptavidin coupled to HRP or AP. This example showsthat if the reporter is an enzyme, it can be the same as, or differentfrom, the enzyme used in the ADEAS.

EXAMPLE 6 Amplification Of Detector Signal In An Assay For HIV p24 WhichUtilizes A Biotinylated Detector Antibody/Streptavidin-HRP DetectionSystem

The Du Pont HIV p24 Antigen ELISA (catalog number NEK 060) was modifiedfor catalyzed reporter deposition as follows: SA-HRP was used at 1/4 theconcentration indicated in the directions. This was followed by a 15min. RT incubation with biotin-tyramine, 10 μg/ml, in 0.1M borate, 0.01%H₂ O₂ pH 8.5 buffer (as in Example 5). Following washing with PBST,SA-HRP at 1/16 the concentration was incubated for 15 min. at RT.Finally, OPD was added as per kit directions. Except for extending thestandard concentrations down to 0.39 pg/ml, no other changes were made.

Results

The results are shown in FIG. 4. This experiment demonstrated that onecan amplify the signal generated by a biotinylated antibody/SA-HRPsystem using catalyzed reporter deposition. Because the concentration ofSA-HRP for both incubations was much less than that for thenon-amplified assay, it was clear that the increased signal wasattributable to reporter deposition and not to a double SA-HRPincubation.

EXAMPLE 7 Reporter Deposition on Membranes

Nitrocellulose (Schleicher & Schuell, BA 85) was spotted with HSVantigen, and then blocked with 1% BSA, 1% non-fat dry milk, in PBSbuffer, overnight. The membranes were incubated for 1 hour at RT withthe analyte dependent enzyme activation system described in Example 1above. The membranes were then incubated with biotin tyramine (fromExample 1) at 2 μl/10 ml 50 mM tris-HCl, 0.01% H₂ O₂, pH 8.0 buffer for15 min. at RT, which was followed by incubation withstreptavidinalkaline phosphatase for 15 min. at RT. Controls were runwhere biotin tyramine was incubated without streptavidin-alkalinephosphatase, and streptavidinalkaline phosphatase was incubated withoutbiotin-tyramine. Visualization of deposited alkaline phosphatase wasfacilitated by the addition of BCIP/NBT (Kirkegaard & Perry). BCIP is5-bromo-4-chloro-indoxyl phosphate and NBT is2,2'-di-(p-nitrophenyl)-5,5'-diphenyl-3,3'-(3,3'-dimethoxy-4,4'-diphenylene)ditetrazoliumchloride. Visualization of the bound anti-HSV-HRP conjugate wasfacilitated by the addition of diaminobenzidine (DAB).

Results

Addition of DAB produced observable brown spots where HSV antigen wasspotted on the nitrocellulose membrane. Addition of BCIP/NBT producedobservable blue spots where HSV antigen was spotted when biotin tyramineand streptavidin-alkaline phosphatase were incubated with the membrane.This showed that alkaline phosphatase was deposited due to HRPactivation of the biotin tyramine conjugate, followed bystreptavidinalkaline phosphatase detection.

EXAMPLE 8 Deposition of Beta-Galactosidase by Horseradish Peroxidase,and Detection by fluorescence

Polystyrene EIA strips (NUNC) were coated with goat anti-mouse IgG (Fcfragment specific) antibody (ICN) in 0.1M carbonate buffer, pH 9.6,overnight at RT. They were then blocked with 2% BSA in carbonate bufferand washed with PBST. Concentrations of mouse IgG in BSA-PBST, as setforth in FIG. 5, were incubated for 1 hour at 37° C. followed by washingwith PBST. Goat antimouse IgG-HRP (ADEAS) purchased from BoehringerMannheim was diluted as recommended by the manufacturer and incubatedfor 1 hour at 37° C. The plate was then washed with PBST. A 1 mg/mlstock solution of BT conjugate (as described in Example 4) in dimethylsulfoxide was prepared, and then added to a 0.1M borate, 0.01% H₂ O₂, pH8.5 buffer at 10 μl/ml (10 μg/ml biotin tyramine). The mixture was addedto the plate and incubated for 15 min. at RT, and then washed with PBST.Streptavidinbeta galactosidase (Bethesda Research Labs) was added andincubated for 15 min. at RT. The assay was also run without catalyzedreporter deposition, i.e., without adding BT. Colorimetric detection ofthe non-amplified assay was achieved after incubation with OPD (forHRP), for 15 min. at RT. Fluorescent detection of the amplified assaywas achieved after the addition of 4-methylumbelliferylbeta-D-galactoside (MUG) (for HRP-beta Gal). Optical densities at 490 nmwere recorded for HRP/OPD on a microtiter plate reader. Fluorescence forHRP-beta Gal/MUG was recorded on a fluorescence microtiter plate reader(Dynatech Laboratories).

Results

The results are shown in FIG. 5. The fluorescent signal was due to thequantitative deposition of biotin tyramine by the HRP ADEAS followed byincubation with streptavidin beta-galactosidase.

EXAMPLE 9 Amplification of a Membrane Assay

Fluorescein-tyramine (FT) was prepared as follows: Solutions of 46.6 mgof 5-(and 6)-carboxyfluorescein succinimidyl ester in 0.3 ml dimethylsulfoxide and 14.6 mg tyramine in 0.3 ml dimethyl sulfoxide wereprepared. Conjugation was achieved by mixing 0.25 ml of each solutionovernight at RT. The solution was used as is.

Three nitrocellulose (Schleicher & Schuell, BA 83) strips were spottedwith 1 μl of mouse IgG at 10 μg/ml, and serial two fold dilutons in PBS.The membranes were blocked with 5% non-fat dry milk in PBST for 30 min,and then washed three times in PBST. A goat anti-mouse IgG-HRP conjugate(Boehringer Mannehim) diluted 1/2000 in 1% BSA-PBST was incubated for 30min. at RT, and the membranes were washed three times with PBST. Thethird membrane was incubated with FT at 20 μg/ml in 0.1M borate, 0.01%H₂ O₂, pH 8.5 buffer for 15 min. at RT, and washed three times in PBST.Then, the second and third membranes were incubated with ananti-fluorescein antibody (Chemicon) which was conjugated to HRP (by theSMCC method of Ishikawa, E., et al., J. Immunoassay, 4, 209-327, 1983)diluted in 1% BSA-PBST for 15 min at RT, and washed three times in PBST.Visualization of all three strips was facilitated by the addition ofdiaminobenzidine for 5 min.

Results

Three spots could be seen on the first two strips indicating a detectionlimit of 2.5 μg/ml, and that the anti-fluorescein-HRP conjugate did notcontribute to additional signal. Six spots could been seen on the thirdstrip indicating a detection limit of 313 ng/ml. The use of thecatalyzed reporter deposition amplification method of the inventionimproved the detection limit of the assay eight fold over that of thenon-amplified assay.

EXAMPLE 10 Preparation of7-(β-Galactosyl)-4-Methylcoumarin-3-Acetamidobiocytin (a)7-Hydroxy-4-methylcoumarin-3-acetic acid, ethyl ester (2)

7-Hydroxy-4-methylcoumarin-3-acetic acid (1) (1 gm, 4.3 mmol) wassuspended in anhydrous ethanol (15 mL), concentrated sulfuric acid (1.5mL) was added and the mixture was heated at reflux for 1 hour. Thereaction mixture was cooled to room temperature, diluted with ethylacetate (100 mL), washed with 5% NaHCO₃ (5×50 mL), water (2×50 mL),saturated NaCl (2×50 mL) dried with sodium sulfate, filtered and thefiltrate was evaporated under reduced pressure to give 1.04 gm (4.0mmol, 92% yield of 2 as a white solid. ¹ H NMR (CDCl₃ /DMSO-d₆): 1.19(3H,t), 2.29 (3H,s), 3.57 (2H,s), 4.06 (2H,q), 6.65 (1H,d), 6.73(1H,dd), 7.44 (1H,d).

TLC: Silica gel, 3% methanol, 1% acetic acid, methylene chloride, R_(f)=0.25.

(b) 7-(Tetraacetyl-β-galactosyl)-4-methylcoumarin-3-acetic acid, ethylester (3)

To a stirred solution of 7-hydroxy-4-methylcoumarin-3-acetic acid, ethylester (2) (1.04 gm, 4.0 mmol), bromo-tetraacetyl-β-galactoside (1.4 gm,3.41 mmol) in acetone (20 mL) was added lN NaOH (3.6 mL) dropwise. Thereaction was stirred at room temperature overnight and TLC (silica gel,3% methanol, 1% acetic acid, methylene chloride) showed about a 50%conversion of starting material to a faster moving spot so an additional0.36 mL of NaOH and 200 mg (0.49 mmol) ofbromo-tetraacetyl-β-galactoside were added and stirring continued for 1hour. TLC showed no change so the reaction mixture was diluted withethyl acetate (100 mL) and washed with water (3×50 mL), saturated sodiumchloride (2×30 mL), dried with sodium sulfate, filtered and the filtratewas evaporated under reduced pressure to give a yellow oil which waspurified by silica gel chromatography eluting with 2% methanol inmethylene chloride. Fractions containing the desired product by TLC werepooled and evaporated to give 1.51 gm of a white foam which wascrystallized from 50% ethanol to give 879 mg (1.51 mmol), 38%) of 3 aswhite needles: m.p. 195°-197° C.

¹ H NMR (CDCl₃): 1.25 (3H,t), 2.0 (3H,s), 2.07 (3H,s), 2.09 (3H,s), 2.17(3H,s), 2.37 (3H,s), 3.7 (2H,s), 4.1 (5H,m), 5.1 (2H,m), 5.5(2H,m), 6.9(2H,m), 7.5 (1H,m).

TLC: Silica gel, 3% methanol, 1% acetic acid, methylene chloride, R_(f)=0.35.

(c) 7-(β-galactosyl)-4-methylcoumarin-3-acetic acid (4)

To a stirred solution of7-(tetraacetyl-β-galactosyl)-4-methylcoumarin-3-acetic acid, ethyl ester(3) (422 mg, 0.712 mmol) in methanol was added 6.8M KOH (5 mL) and thesolution was stirred overnight at room temperature. The solution wasneutralized by the addition of 1N HCl and evaporated to dryness underreduced pressure. The residue was dissolved in water (10 mL) anddesalted using C₁₈ Sep Pack Cartridges in 10×1 mL portions. The C₁₈ Seppack Cartridge (Waters Part No. 65910) was washed with methanol (3 mL)and water (3 mL) before the sample was applied in 1 mL. The cartridgewas then washed with water (3 mL) to remove any salt followed bymetahnol (3 mL) which eluted the desired product as determined by TLC(silica gel, acetone:20 mM NaOAc 9:1). The methanol washes were pooledand evaporated under reduced pressure and the while solid obtained wasrecrystallized from ethanol/water to give 170 mg (0.43 mmol, 60%) of 4as white crystals: m.p. 186°-188° C.

¹ H NMR (DMSO-d₆) 2.37 (3H,s), 3.4-3.7 (9H,m), 4.6 (1H,br), 4.7 (1H,br),5.0 (1H,d), 5.3 (1H,br), 7.0 (2H,m), 7.7 (1H,m).

TLC: Silica gel, acetone:20mM NaOAc 9:1, R_(f) =0.7.

(d) 7-(β-galactosyl)-4-methylcoumarin-3-acetamidobiocytin (5)

To a stirred solution of 7-(β-galactosyl)-4-methylcoumarin-3-acetic acid(4) (146 mg, 0.368 mmol) in DMF (3 mL) was added N-hydroxysuccinimide(46 mg, 0.400 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (74 mg, 0.386 mmol). The solution was stirred at roomtemperature for 2.5 hours and added dropwise to a solution of biocytin(250 mg, 0.671 mmol) in 0.1M NaHCO₃ (5 mL) and DMF (1 mL) and stirred atroom temperature for 2 hours. The solution was evaporated to drynessunder reduced pressure and the residue was washed with hot methanol(3×10 mL). The methanol washes were evaporated to give an off whitesolid which was dissolved in water (3 mL) and purified by preparativeHPLC (4×0.75 mL Injections) on ZORBAX C8 (4.6 mm ×25 cm) eluting with alinear gradient of 5-15% acetonitrile in 0.1M triethylammonium acetatepH=7 at 2 mL/min. The desired product eluted at the end of the gradientas a single peak which was collected, evaporated, stripped from water(3×5 mL) to give 54 mg (0.07 mmol, 20%) of 5 as a white solid.

¹ H NMR (DMSO-d₆) contains peaks which correspond to biocytin and7-(β-galactosyl)-4-methylcoumarin-3-acetic acid.

TLC: Silica gel, methanol, R_(f) =0.7

The synthesis of this compound is illustrated in FIG. 6.

EXAMPLE 11 Amplification Using a β-Galactosidase ADEAS

The preparation of 7-(β-galactosyl)-4-methylcoumarin-3-acetamidobiocytin(GCB) was described in Example 10 above.

Monoclonal antibodies were prepared by immunizing mice with a7-hydroxy-4-methylcoumarin-3-acetic acid-BSA conjugate which wassynthesized by reacting 7-hydroxy-4-methylcoumarin-3-acetic acid,succinimidyl ester (Molecular Probes, Eugene, OR) with BSA. Hybridomaswere produced by fusion of lymph node lymphocytes (Mirza, I. H. et al.,J. Immunol. Methods 105, 235-243, 1987) by the method of de St. Groth,F. et al., J. Immunol. Methods 35, 1, 1980.

The assay was performed as follows: Polystyrene EIA strips (NUNC) werecoated with goat anti-mouse IgG (Fc fragment specific) antibody (ICN) in0.1M carbonate buffer pH 9.6, overnight at RT. The strips were thenblocked with 2% BSA in PBS. After washing with PBST, theabove-identified hydridoma supernatant diluted 1/5 in 1% BSA-PBST wasincubated with the strips for 2 hr at RT. The strips were washed again.β-Galactosidase at concentrations ranging from 0.01 to 50 ng/ml (plus a0 ng/ml blank) in 10 mM sodium phosphate, 1 mM MgCl₂, 0.1% BSA, pH 7.0was added to the wells with either o-nitrophenyl-β-D-galactoside (ONPG)at 1 mg/ml (for colorimetric detection of β-galactosidase activitywithout amplification) or GCB at 100 ng/ml (for amplification) for 30min at RT. The absorbance in the wells containing ONPG was read at 405nm. The wells containing GCB were washed with PBST, and incubated withstreptavidin-HRP for 15 min at RT. Following washing with PBST, the HRPsubstrate tetramethylbenzidine (SOMA Labs, Romeo, Mich.) was added for30 min at RT, the reaction stopped with 3N H₂ SO_(4/1) N HCl, and theabsorbance at 450 nm determined.

Results

The results are shown in FIG. 7. The net absorbance (blank subtracted)is plotted vs. the β-galactosidase concentration. A 64-fold improvementin β-galactosidase detection was obtained using the catalyzed reporterdeposition amplification method when compared to direct colorimetricdetection.

What is claimed is:
 1. A method for the detection or quantitation of ananalyte in an assay which comprises using an analyte dependent enzymeactivation system comprising at least one enzyme to react with aconjugate consisting of a detectably labeled substrate specific for theenzyme system to form an activated conjugate which depositssubstantially wherever at least one receptor for the activated conjugateis immobilized, said receptor not being reactive with the analytedependent enzyme activation system, wherein deposited detectable labelseither directly or indirectly generate a signal which can be detected orquantitated.
 2. A method according to claim 1 wherein at least oneenzyme of the analyte dependent enzyme activation system is selectedfrom the group consisting of oxidoreductases, hydrolases, lyases,transferases, isomerases, and ligases.
 3. A method according to claim 2wherein the enzyme is selected from the group consisting of peroxidases,oxidases, phosphatases, esterases and glycosidases.
 4. A methodaccording to claim 3 wherein the enzyme is selected from the groupconsisting of horseradish peroxidase, glucose oxidase, alkalinephosphatase and beta-galactosidase.
 5. A method according to claim 4wherein the enzyme is horseradish peroxidase.
 6. A method according toclaim 5 wherein the conjugate is selected from the group consisting ofbiotin-tyramine, p=hydroxyphenylpropionylbiocytin, orfluorescein-tyramine.
 7. A method according to claim 1 wherein theconjugate is reacted with detectably labeled antibody.
 8. A methodaccording to claim 1 wherein the conjugate is reacted with a detectablylabeled member of a specific binding pair.
 9. A method according toclaim 1 wherein the conjugate is reacted with detectably labeledstreptavidin.
 10. A method according to claim 1 wherein the detectablelabel is selected from the group consisting of enzymes, radioactiveisotopes, fluorogenic, chemiluminescent, or electrochemical materials ora member of a specific binding pair.